Method for transfer of freeze-dried specimens

ABSTRACT

A method and apparatus for transferring freeze-dried sections of biological material from a freeze-drier to an electron microscope or to an apparatus for applying a hydrophobic coating to the sections. The sections are transferred in an evacuated container to prevent rehydration of the sections during the transfer.

United States Patent Hodson et al.

[451 May 27, 1975 [56] References Cited UNITED STATES PATENTS 3.42l,229 1/1969 Kniese .r 34/5 X Primary Examiner-lohn Jr Camby Attorney, Agent. or Firm-Browdy and Neimark [57] ABSTRACT A method and apparatus for transferring freeze-dried sections of biological material from a freeze-drier to an electron microscope or to an apparatus for applying a hydrophobic coating to the sections. The sections are transferred in an evacuated container to prevent rehydration of the sections during the transfer.

4 Claims, 5 Drawing Figures /PATENTED W2 7 I975 SHEET D38F 3 METHOD FOR TRANSFER OF FREEZE-DRIED SPECIMENS This invention relates to the transfer of freeze-dried specimens from one apparatus to another.

The invention is concerned in particular with the transfer of freeze-dried biological sections from a microtome to other apparatus There have in recent years been developed ultramicrotomes capable of cutting thin sections of frozen biological tissue for inspection by an electron microscope. lt is necessary for the examination of the section under an electron microscope that the section be dehydrated before its temperature is allowed to rise, i.e. for the section to be freezedried. A problem has arisen in the transfer of such freeze-dried sections from the ultramicrotome to the electron microscope or other apparatus in which the section is to be further treated, in that the section tends rapidly to absorb water from the atmosphere as soon as it is removed from the freeze-drier.

It is an object of the present invention to provide a method of transferring freeze-dried specimens from one apparatus to another whilst preventing rehydration of the specimens.

This invention includes the method of transferring freeze-dried specimens from a first apparatus to an evacuated chamber of a second apparatus, comprising the steps of cooling the interior of an air-tight transfer container, connecting the container to the first apparatus so that a frozen specimen in the first apparatus can be inserted into the container, evacuating the interior of the container, sealing the container after insertion into it of the frozen specimen, transferring the container to the second apparatus and effecting communication between the interior of the container and the evacuated chamber of the second apparatus.

Preferably, the transfer container is evacuated after insertion into it of the specimen, thereby dehydrating the specimen.

The invention also includes a container for transfer ring freeze-dried specimens from a first apparatus to an evacuated chamber of a second apparatus comprising a container body adapted to accommodate at least one freeze-dried specimen, valve means through which the interior of the body can be evacuated, aperture means in the body through which the specimen can be inserted into and withdrawn from the container, and closure means for closing the aperture means so as to maintain a vacuum in the container as it is transferred from the first apparatus to the second apparatus.

The invention also includes the method of protecting a freeze-dried specimen from rehydration, comprising coating the entire surface area of the freeze-dried specimen in vacuo with a layer of carbon or other hydrophobic layer.

The invention will now be described, by way of examplc, with reference to the accompanying drawings, in which:

FIG. 1 is a side elevation of a transfer container constructed in accordance with the invention,

FIG. 2 is an underneath plan view of the container of FIG. 1,

FIG. 3 is a section taken on line lll-lll of FIG. 1, showing the container in position on a mounting block,

FIG. 4 is a diagrammatic plan view of part ofa microtome showing the container of FIGS. l to 3 in the lowtemperature box of the microtome, and

FIG. 5 is a diagrammatic side elevation, partly in section, ofa bell jar containing equipment for carbon coating specimens in the container of FIGS. 1 to 3.

FIGS. 1 to 4 show a transfer container 10 used to transfer freeze dried specimens from an ultramicrotome to an evacuated bell jar containing apparatus for applying a hydrophobic coating to the specimens. The transfer container comprises a generally cylindrical body 12 of steel or other suitable material, formed with a cylindrical bore 14. At one end an enlarged part 16 of the body is formed with a counter-bore 18 which is connected to bore 16 by a step 20. Step 20 provides an annular platform which supports a plate 2! formed with a number of bores 23 each of which can receive a carbon-coated copper grid of known form on which can be mounted a section cut by the ultramicrotomc. The counter-bore 18 opens into a recess 22 into which fits a disc-like cap 24, an O-ring sea] 26 being interposed between cap 24 and the bottom wall of recess 22. The cap 24 is formed wholly or partly of ferromagnetic material to enable it to be manipulated by means of a magnet.

At the lower end of body 12, a small diameter bore 28 connects the bore 14 to a counter-bore 30 containing a one-way valve 32. Valve 32 consists of a valve member 34 seating against an O-ring seal 36 fitted in a groove in the axially facing wall of counter-bore 30, and biased against the O-ring by a helical compression spring 38 acting between the valve member and a plug 40, which is secured to body 12 by means of an external screw-thread engaging an internal thread in counterbore 30. The plug 40 has a bore 42 communicating with the counter-bore 30. Projecting a short way from the lower face 44 of the body 12 is an externally screwthreaded spigot 46, the counter-bore 30 extending through the spigot.

The transfer container 10 can be attached to a mounting block 50 by means ofthe spigot 46 which can be screwed into an internally screw-threaded cylindrical recess 52 in block 50. Leading from recess 52 is a bore 54 through which air can be drawn from container 10 when it is fitted to block 50. The upper face of block 50 is formed with an annular groove 56 accommodating an O-ring 58 which provides a seal between the mating faces of container 10 and block 50.

The mounting block 50 is fixed inside a box 60 surrounding the knife 62 and specimen holder 64 of an ultramicrotome (see FIG. 4). The box 60 is provided with cooling means so that the microtome can be used for cutting thin selections from a frozen specimen 66, e.g. of biological tissue, held by specimen holder 64. The ultramicrotome may be similar to that described in copending patent applications No. 49021/68 (published as specification No. 1,307,974) and No. 16410/70 and in the paper entitled Ultracryotorny: a technique for cutting ultrathin sections of unfixed frozen biological tissues for electron microscopy" by Hodson and Marshall, Journal of Microscopy, Volume 91, Part 2, April 1970, pages l05l 17. The block 50 is fixed adjacent to one wall 68 of box 60, and a connector 70 extends through wall 68 to the bore 52 in block 50, the connector enabling the bore to be connected to a vacuum line.

In use, the transfer container 10 is screwed onto mounting block 50in box 60, with the cap 24 removed, as shown in FIG. 4. The specimen holder 64 and knife 62 are cooled to a sufficiently low temperature, as described in the references cited above, and a frozen specimen is fixed to the specimen holder. The mounting block 50 and container 10 are similarly cooled to a low temperature.

One or more thin sections cut by the knife 62 from specimen 66 are mounted on carbon-coated grids and the grids are positioned on plate 21 in container 10, using suitable manipulating tweezers, and the cap 24 is placed in position on container 10. The interior of container 10 is then evacuated, through bores 28 and 42, bore 54 in block 50, and connector 70, the pressure in container 10 being reduced sufficiently, say to 10 torr, to effect dehydration of each section whilst it remains frozen. When the vacuum is cut off, valve member 34 is held by atmospheric pressure against its seating, and the cap 24 is held in position by atmospheric pressure, the O-ring seals 26 and 36 providing air-tight seals, so that the container 10 can be unscrewed from mounting block 50 whilst retaining its vacuum The container 10 is then removed from box 60 and the temperature of the container and the sections in it is allowed to rise to room temperature. Having freeze-dried the sections, the container 10 can be removed from the box and transferred to another instrument without danger of rehydration of the section.

The transfer container 10 is removed from the box 60 and positioned in a vacuum bell jar 72 (FIG. containing equipment for carbon coating the sections. The container is supported in the bell jar on a plate 74 having a screw-threaded recess to receive spigot 46 on container 10, and rotatably mounted in a fixed support 76. The plate 74 can be rotated about an axis inclined at 30 to the vertical by means of an electric motor 78 driving the plate through a sprocket and chain connection 80, a shaft 82 extending into the bell jar through a vacuum-tight seal 84, and an angled connection 86. The bell jar contains equipment of known form for providing a coating of carbon on a specimen, the equipment consisting of a pair of carbon electrodes 90 and 92 positioned so that an are formed between the electrodes by passage of an electric current is located vertically above the centre of plate 21 in container 10.

After the transfer container has been positioned in the bell jar 72, the bell jar is evacuated to a pressure equal to the pressure in the container, so that the cap 24 is released from the body of the container. The cap 24 is removed by means ofa magnet manipulated from outside the bell jar. Current is then passed between the electrodes 90 and 92 to produce an are which causes carbon atoms to be deposited on the grid-mounted sections on plate 21, whilst the container 10 is rotated about its axis to ensure that the entire exposed surface area of each section is coated with a layer of carbon. The vacuum in the bell jar is then released and the container removed from the bell jar so that the sections can be taken from it.

It has been found that a carbon coating applied to freeze-dried sections of biological material acts as a hydrophobic barrier, so that the sections can be handled normally, with no danger of rehydration, as they are transferred to an electron microscope. The carbon coating does not appreciably affect the transmissivity of the section to high energy electrons and therefore does not interfere with the examination of the section under the electron microscope. Indeed, as is well known, the presence of a conducting layer on specimens can facilitate their examination under an electron microscope.

lt will be apparent that various modifications could be made in the described method. For example, the frozen sections might be dehydrated in a freeze-drying chamber in the ultramicrotome casing before the transfer container is evacuated and the sections are inserted into the container. The transfer container could be modified to enable the freeze-dried sections to be transferred direct from the container into an electron microscope, without the need for the intermediate stage of forming a hydrophobic coating on the sections.

We claim:

1. The method of transferring freeze-dried specimens from a first apparatus to an evacuated chamber of a second apparatus comprising:

placing in communication with the casing of a first apparatus an air-tight transfer container separable from and movable independently of said first and second apparatus, said first apparatus including a casing, a specimen holder and a microtome having a knife operable to cut thin sections from a frozen specimen of biological material held in said specimen holder, said casing surrounding said knife and specimen holder;

cutting thin sections from said frozen specimen of hiological material;

cooling the interior of the transfer container;

inserting into the transfer container sections of said frozen specimen from said first apparatus; evacuating the interior of said transfer container; sealing the transfer container;

removing the transfer container from the first apparatus; and

transferring the transfer container to the second apparatus and effecting communication between the interior of the transfer container and the evacuated chamber of the second apparatus.

2. The method claimed in claim 1 wherein the step of evacuating the interior of the transfer container after insertion into it of the frozen specimen, thereby to dehydrate the specimen.

3. The method as claimed in claim 1 wherein the step of dehydrating the frozen specimen and evacuating the transfer container occurs before inserting the specimen into the transfer container.

4. The method claimed in claim 1, including transferring the container to chamber containing apparatus for carbon coating, evacuating the chamber, opening the container and coating with a layer of carbon the entire exposed surface of the or each section held in the container. 

1. The method of transferring freeze-dried specimens from a first apparatus to an evacuated chamber of a second apparatus comprising: placing in communication with the casing of a first apparatus an air-tight transfer container separable from and movable independently of said first and second apparatus, said first apparatus including a casing, a specimen holdeR and a microtome having a knife operable to cut thin sections from a frozen specimen of biological material held in said specimen holder, said casing surrounding said knife and specimen holder; cutting thin sections from said frozen specimen of biological material; cooling the interior of the transfer container; inserting into the transfer container sections of said frozen specimen from said first apparatus; evacuating the interior of said transfer container; sealing the transfer container; removing the transfer container from the first apparatus; and transferring the transfer container to the second apparatus and effecting communication between the interior of the transfer container and the evacuated chamber of the second apparatus.
 2. The method claimed in claim 1 wherein the step of evacuating the interior of the transfer container after insertion into it of the frozen specimen, thereby to dehydrate the specimen.
 3. The method as claimed in claim 1 wherein the step of dehydrating the frozen specimen and evacuating the transfer container occurs before inserting the specimen into the transfer container.
 4. The method claimed in claim 1, including transferring the container to chamber containing apparatus for carbon coating, evacuating the chamber, opening the container and coating with a layer of carbon the entire exposed surface of the or each section held in the container. 